CN112816537A - Protein molecular imprinting electrochemical sensor based on temperature-sensitive sodium alginate self-assembled micelle and preparation method and application thereof - Google Patents
Protein molecular imprinting electrochemical sensor based on temperature-sensitive sodium alginate self-assembled micelle and preparation method and application thereof Download PDFInfo
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- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 3
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- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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Abstract
The invention provides a protein molecular imprinting electrochemical sensor based on a temperature-sensitive sodium alginate self-assembled micelle, a preparation method and an application thereof2And (3) crosslinking in an aqueous solution, fixing binding sites, drying in the air, and eluting in a mixed solution of methanol and acetic acid to remove template proteins to obtain the protein molecular imprinting electrochemical sensor. The prepared protein molecular imprinting sensor has high sensitivity, low detection limit, low price and convenient operation, and has potential application in the aspects of environmental detection, drug analysis, food safety and the likeAnd (4) foreground.
Description
Technical Field
The invention belongs to an electrochemical sensor, and particularly relates to a protein molecular imprinting electrochemical sensor based on a temperature-sensitive sodium alginate self-assembled micelle, and a preparation method and application thereof.
Background
Proteomics is one of the most important research fields at present, and has a very close relationship with human health and social development. The deep research on the protein not only can provide a material basis for disclosing the life activity rule, but also can provide a theoretical basis and a solution way for the explanation and attack of a plurality of disease mechanisms. In proteomics research, there is a need to develop methods for selectively isolating, identifying and detecting specific target proteins from complex matrix samples. Therefore, the development of a new separation and identification method will play an important role in the field of proteomics.
The western blotting technique is a novel technique developed by integrating a plurality of disciplines such as macromolecule synthesis, protein molecule identification, bionic bioengineering and the like, and is an effective method for selectively separating and identifying target protein from a complex matrix sample. However, mass transfer and diffusion in imprinted polymers are difficult due to the large spatial size of the protein molecules. The macromolecular self-assembly micelle with the nano structure is favorable for improving the mass transfer rate of protein in the imprinted polymer micelle, but due to the characteristic of compact structure, the template in the macromolecular self-assembly micelle ensures that the mass transfer diffusion resistance of the template protein is larger, so that the elution and recombination are not facilitated, and the further improvement of the identification and detection performance of the protein molecular imprinting sensor based on the macromolecular self-assembly micelle is limited.
The temperature-sensitive molecularly imprinted polymer is the most promising protein imprinted material at present, can generate continuous and reversible swelling and shrinking under the stimulation of temperature, and promotes the change of the distance and the relative position between an imprinted functional group and template protein, thereby being beneficial to separating the template protein from the polymer to form the molecularly imprinted polymer or recombining the molecularly imprinted polymer to realize specific adsorption, effectively regulating the combined amount of the molecularly imprinted polymer and the template protein, and improving the adsorption capacity and the recognition detection performance of the molecularly imprinted polymer.
Disclosure of Invention
The invention aims to provide a protein molecular imprinting electrochemical sensor based on a temperature-sensitive sodium alginate self-assembled micelle and a preparation method thereof.
The invention also aims to provide the application of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle, which is used for detecting protein, has the advantages of high detection sensitivity, low price, convenient operation and potential application prospect in the aspects of environmental detection, drug analysis, food safety and the like.
The specific technical scheme of the invention is as follows:
the preparation method of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle comprises the following steps:
1) preparing temperature-sensitive sodium alginate;
2) preparing a protein/temperature sensitive sodium alginate micelle;
3) a protein/temperature sensitive sodium alginate micelle modified electrode;
4) and (3) preparing the protein molecular imprinting electrochemical sensor.
Further, in the step 1), the method for preparing the temperature-sensitive sodium alginate comprises the following steps:
placing sodium alginate in water, stirring for dissolving, removing oxygen in water, adding initiator and N-isopropyl acrylamide monomer, and sealing for reaction; and after the reaction is finished, standing, naturally cooling, purifying and drying to obtain the thermo-sensitive sodium alginate.
The water in step 1) is preferably distilled water.
In the step 1), sodium alginate is put into water and stirred to be dissolved, and the concentration of a sodium alginate repeating unit in the obtained sodium alginate water solution is 0.01-10 mol/L.
In the step 1), the initiator is ammonium ceric nitrate, azodiisobutyronitrile or potassium persulfate; the mass ratio of the initiator to the sodium alginate repeating units is 1:20-1: 2.
In the step 1), the mass ratio of the sodium alginate repeating unit to the N-isopropylacrylamide monomer is 1:10-10: 1.
In the step 1), the reaction is carried out at the reaction temperature of 10-80 ℃ for 1-15 h.
The purification in the step 1) is purification by dialysis, reactants are filled into a dialysis bag MW8000-10000, and the dialysis is carried out by using distilled water.
The drying refers to freeze drying at-70 deg.C for 24-72 h.
The step 2) is specifically as follows: dissolving the temperature-sensitive sodium alginate prepared in the step 1) in water to obtain a temperature-sensitive sodium alginate solution, dropwise adding a phosphate buffer solution of protein into the temperature-sensitive sodium alginate solution, and self-assembling the temperature-sensitive sodium alginate solution into the protein/temperature-sensitive sodium alginate micelle through weak interaction force between the temperature-sensitive sodium alginate solution and the protein buffer solution.
Further, during the addition, the next drop is added after the previous drop of phosphate buffered solution of protein is completely dispersed in the solution.
The water in step 2) is preferably distilled water.
The protein in the step 2) is bovine serum albumin, bovine hemoglobin, lysozyme, trypsin, horseradish peroxidase or glucolase.
The preparation method of the phosphate buffer solution of the protein in the step 2) comprises the following steps: dissolving the protein in a phosphate buffer solution; the concentration of the protein in the phosphate buffer solution of the obtained protein is 0.001-100 mg/mL. The concentration of the phosphate buffer solution is 0.01-1.0mol/L, and the pH value is 3.0-10.0.
Dissolving the temperature-sensitive sodium alginate in the step 2) into water, wherein the concentration of the obtained temperature-sensitive sodium alginate solution is 0.001-100 mg/mL;
the volume ratio of the temperature-sensitive sodium alginate solution in the step 2) to the phosphate buffer solution of the protein is 10:1-1: 10.
The step 3) is specifically as follows: and modifying the protein/temperature sensitive sodium alginate micelle solution on the surface of the electrode, and drying to obtain the protein/temperature sensitive sodium alginate micelle modified electrode.
The electrode is a gold electrode or a glassy carbon electrode;
before the electrode is used, the following treatments are carried out: the electrodes were sequentially coated with 0.3 μm and 0.05 μm of α -Al2O3And (3) polishing the surface of the powder, cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol respectively, and naturally drying the powder.
The concentration of the protein/temperature sensitive sodium alginate micelle solution in the step 3) is 0.001-100mg/mL, and the protein/temperature sensitive sodium alginate micelle solution is modified to the surface of the electrode, and the volume is 1-20 muL.
Drying in the step 3) at the temperature of 20-60 ℃ for 10-30 h.
The step 4) is specifically as follows: soaking the modified electrode prepared in the step 3) in a calcium ion solution by using Ca2+Fixing the binding site of the template protein and the temperature sensitive sodium alginate under the cross-linking action of the sodium alginate, taking out and drying; eluting to remove the template protein, and drying at room temperature to obtain the protein molecular imprinting electrochemical sensor.
Soaking the modified electrode prepared in the step 3) in a calcium ion solution at 20-40 ℃ for 1-2 h.
The concentration of the calcium ion solution in the step 4) is 0.001-100 mg/mL; preferably CaCl2And (3) solution.
Step 4), drying refers to drying at the temperature of 20-60 ℃ for 20-30 h;
and 4) eluting in the step 4), wherein the eluent is a mixed solution of methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is 10:1-1: 10.
The protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle is prepared by the method.
The invention provides an application of a protein molecular imprinting electrochemical sensor based on a temperature-sensitive sodium alginate self-assembled micelle, which is used for detecting protein. The specific detection method comprises the following steps:
s1, placing the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle in a phosphate solution (blank solution) with the protein concentration of 0, standing and adsorbing, placing in an electrolyte solution, and measuring the electrochemical response value of the blank solution by using a differential conventional pulse voltammetry;
s2, placing the molecularly imprinted electrochemical sensor in phosphate buffer solution of protein with certain concentration, placing the molecularly imprinted electrochemical sensor in electrolyte solution after standing and adsorption, measuring an electrochemical response value by using a differential conventional pulse voltammetry method, and taking out the sensor to elute the protein;
s3, placing the eluted molecularly imprinted electrochemical sensor in a phosphate buffer solution of a protein solution with another concentration, and repeating the step S2;
s4, constructing a linear relation by taking the absolute value of the difference between the electrochemical response value measured under a certain protein concentration and the electrochemical response value measured by the blank solution as a ordinate and the protein concentration as an abscissa.
Further, the pH value of the phosphate buffer solution is 7.2, and the concentration is 0.1 mol/L; differential conventional pulse voltammetry (DNPV) measurements, scan ranging from 0.1V to 1.0V; the scanning rate is 20 mV/s; the electrolyte is a phosphate buffer solution with pH of 7.2 and concentration of 0.1mol/L, which contains 5mmol/L potassium ferricyanide, 5mmol/L potassium ferrocyanide and 0.1mol/L potassium chloride.
And respectively placing the prepared electrochemical sensors in a blank solution and a protein solution with unknown concentration, respectively measuring electrochemical response values of the two electrochemical sensors by using a differential conventional pulse voltammetry method, calculating an absolute value of a difference value, and obtaining the protein concentration according to the obtained absolute value of the difference value and the linear relation.
The poly N-isopropyl acrylamide (PNIPAAm) macromolecular chain has hydrophilic amide groups and hydrophobic isopropyl groups, and has a temperature-sensitive polymer. The minimum critical solution temperature (LCST) of PNIPAAm is 35 ℃. The strong interaction of water molecules with the hydrogen bonds of the PNIPAAm group makes it soluble in water when the temperature is below LCST; when the temperature is higher than the LCST, the interaction between hydrogen bonds is broken and the groups are made hydrophobic, PNIPAAm undergoes volume shrinkage in aqueous solution. Due to the temperature-sensitive characteristic of PNIPAAm, the PNIPAAm has wide application prospect in the field of preparation of molecularly imprinted polymers.
The invention discloses a method for preparing a temperature-sensitive sodium alginate polymer by polymerizing an N-isopropylacrylamide (NIPAM) monomer to a sodium alginate side chain through free radical initiation, self-assembling the protein/temperature-sensitive sodium alginate micelle in a solution by utilizing weak interaction force between protein and temperature-sensitive sodium alginate, modifying the protein/temperature-sensitive sodium alginate micelle to the surface of an electrode, airing the electrode, and placing the electrode on CaCl2And (3) crosslinking in an aqueous solution, fixing binding sites, drying in the air, and eluting in a mixed solution of methanol and acetic acid to remove template proteins to obtain the protein molecular imprinting electrochemical sensor. The prepared protein molecular imprinting sensor has the advantages of high sensitivity, low detection limit, low price and convenient operation, and has potential application prospects in the aspects of environmental detection, drug analysis, food safety and the like.
The main advantages of the invention are:
(1) the protein/temperature sensitive sodium alginate micelle used in the invention has good biocompatibility and is beneficial to the maintenance of protein activity in the preparation process of the sensor.
(2) The temperature-sensitive protein molecular imprinting electrochemical sensor prepared by the invention can optimize the detection performance by changing the environmental temperature.
(3) The temperature-sensitive protein molecular imprinting electrochemical sensor prepared by the invention has the advantages of high detection sensitivity, wide linear range, quick and simple reading, low cost and the like.
(4) The invention combines the temperature-sensitive sodium alginate self-assembly technology, the protein molecular imprinting technology and the electrochemical biosensor to prepare the protein molecular imprinting sensor, and has potential application prospect in the fields of environmental detection, drug analysis, food safety and the like.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) image of the BSA/temperature sensitive sodium alginate micelle in example 1;
FIG. 2 is a cyclic voltammetry spectrogram of different modification stages of a preparation process of a protein molecular imprinting sensor;
FIG. 3 shows the differential conventional pulse voltammetry curve (a) and the linear curve (b) of the Western blot sensor in different concentrations of protein solution.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
The preparation method of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle comprises the following steps:
1) preparing temperature-sensitive sodium alginate: placing 1g of sodium alginate (SA, the molar weight of a sodium alginate repeating unit is 176) in a three-neck flask containing 100mL of distilled water, stirring and dissolving at room temperature, and introducing nitrogen gas to remove oxygen dissolved in the solvent; adding 0.5g of ammonium ceric nitrate and 3g N-isopropyl acrylamide (NIPAAm) monomer, heating to 40 ℃ in a water bath, and carrying out a closed reaction for 6 hours; after the reaction is finished, standing, naturally cooling, and putting the reactant into a dialysis bag MW8000-10000 for dialysis and purification. After purification, the sample is frozen and dried at the temperature of 70 ℃ below zero for 72 hours to obtain the temperature sensitive sodium alginate (SA-g-PNIPAAm) copolymer.
2) Preparing a protein/temperature sensitive sodium alginate micelle: dissolving bovine serum albumin in a phosphate buffer solution with the pH of 7.2 and the concentration of 0.1mol/L to obtain 0.25mg/mL of bovine serum albumin solution, dissolving temperature-sensitive sodium alginate in distilled water to prepare 0.05mg/mL of solution, taking 1mL of temperature-sensitive sodium alginate solution, dropwise adding 200 mu L of 0.25g/L of bovine serum albumin solution, adding the next drop of solution when one drop of bovine serum albumin solution is completely dispersed in the solution, and self-assembling the bovine serum albumin/temperature-sensitive sodium alginate (BSA/SA-g-PNIPAAM) micelle through weak interaction force between the two under magnetic stirring.
FIG. 1 is a Scanning Electron Microscope (SEM) image of BSA/SA-g-PNIPAAM micelles in example 1. As can be seen from the figure, BSA/SA-g-PNIPAAm is a spherical aggregate with a particle size of about 1 μm. A sample detection step: 0.1mg/mL BSA/SA-g-PNIPAAM micellar aqueous dispersion was dropped on a glass slide, dried in an oven at 40 ℃ and characterized by SEM.
3) Protein/temperature sensitive sodium alginate micelle modified electrode: the bare gold electrode was sequentially coated with 0.3 μm and 0.05 μm of alpha-Al2O3And (3) polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally drying the powder, modifying 5 mu L of 0.05mg/mL protein/temperature-sensitive sodium alginate micelle on the surface of the electrode at the temperature of 20 ℃, and drying the modified electrode for 24 hours to obtain the protein/temperature-sensitive sodium alginate micelle modified electrode.
4) Preparation of a protein molecular imprinting electrochemical sensor: soaking the dried protein/temperature-sensitive sodium alginate micelle modified electrode in 0.5mg/mL CaCl2In the aqueous solution, Ca is utilized2+Fixing the binding site of the template protein and the temperature sensitive sodium alginate under the cross-linking action of the sodium alginate, soaking for 2h at 20 ℃, and then drying in an oven at 40 ℃ for 24 h; placing in eluent (V) at room temperatureMethanol:VAcetic acidAnd (1: 6) eluting to remove the template protein, and drying at room temperature to obtain the protein molecular imprinting electrochemical sensor.
FIG. 2 is a cyclic voltammogram of different modification stages of the preparation process of the Western blot sensor of example 1. CV conditions: scanning range from-0.8V to 0.2V; the scan rate was 100 mV/s. The electrolyte is a phosphate buffer solution containing potassium ferricyanide (5mmol/L), potassium ferrocyanide (5mmol/L) and potassium chloride (0.1mol/L), and having a pH of 7.2 and a concentration of 0.1 mol/L. Example 1, step 3) of bare electrode cyclic voltammetry curve corresponds to the bare gold electrode curve in fig. 2, example 1, step 3) of protein/temperature sensitive sodium alginate micelle modified electrode cyclic voltammetry curve prepared corresponds to the micelle modified electrode curve in fig. 2, and example 1, step 4) of Ca in example 12+The cyclic voltammogram of the crosslinked electrode corresponds to the crosslinked curve in FIG. 2; example 1 the cyclic voltammogram of the electrode after elution in step 4) corresponds to the post-electroelution curve in figure 1; the adsorption curve in FIG. 2 corresponds to the adsorption curve after the adsorption when the molecular imprinting sensor detects bovine serum albumin. The cyclic voltammetry curve of the bare electrode in the electrolyte solution has a pair of symmetrical redox peaks, which shows that the conductivity is good; the oxidation-reduction peak of the cyclic voltammetry curve of the BSA/SA-g-PNIPAAM modified electrode in an electrolyte solution is reduced, which is caused by an insulating BSA/SA-g-PNIPAAM micelle; ca by2+After crosslinking, the redox peak of the cyclic voltammogram was further reduced, indicating the formation of a crosslinked network; after the template molecules are removed by elution, the formed imprinted cavity is beneficial to the entry and exit of small molecules, so that the oxidation-reduction peak of the cyclic voltammetry graph is slightly increased; after adsorbing the template bovine serum albumin, the blotting cavity was filled with bovine serum albumin, and the redox peak of the cyclic voltammogram was reduced.
The method for detecting the bovine serum albumin by using the prepared sensor comprises the following steps: the sensors are respectively arranged at 0 and 1 multiplied by 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、1×10-3In the phosphate buffer solution of bovine serum albumin with different mol/L concentrations, wherein the pH value of the phosphate buffer solution is 7.2, the concentration is 0.1mol/L, and standing and adsorbing are carried out for 5 min. Placing in electrolyte solution, measuring electrochemical response value by differential conventional pulse voltammetry, scanning range is from 0.1V to 1.0V, scanning speed is 20mV/s, electrolyte is phosphate buffer solution with pH value of 7.2 and concentration of 0.1mol/L containing 5mmol/L potassium ferricyanide, 5mmol/L potassium ferrocyanide and 0.1mol/L potassium chloride. And taking out the solution after each measurement to elute bovine serum albumin for 3 min, detecting the next concentration, and constructing a linear relation by taking the absolute value of the difference value between the electrochemical response value measured under a certain concentration and the electrochemical response value measured by the blank solution as a vertical coordinate and the protein concentration as a horizontal coordinate. The linear equation is Δ I-17.614 +1.601lg (C)BSA) Δ I is the current difference in units of μ A, CBSAThe concentration of bovine serum albumin is expressed in mol/L; the square of the correlation coefficient was 0.996. The linear relationship is shown in fig. 3.
Example 2
The preparation method of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle comprises the following steps:
1) preparation of thermo-sensitive sodium alginate
Placing 1g of sodium alginate in a three-neck flask containing 100mL of distilled water, stirring and dissolving at room temperature, and introducing nitrogen gas to remove oxygen dissolved in the solvent; adding 0.4g of ammonium ceric nitrate and 4g N-isopropyl acrylamide monomer, heating to 50 ℃ in a water bath, and carrying out a closed reaction for 5 hours; and after the reaction is finished, standing, naturally cooling, purifying and drying to obtain the thermo-sensitive sodium alginate.
2) Preparation of protein/temperature-sensitive sodium alginate micelle
Dissolving temperature-sensitive sodium alginate in distilled water to prepare 0.2mg/mL solution, taking 1mL, dropwise adding 200 mu L of 0.2mg/mL bovine serum albumin solution, and self-assembling the protein/temperature-sensitive sodium alginate micelle through weak interaction force between the two under magnetic stirring.
3) Preparation of protein/temperature-sensitive sodium alginate micelle modified electrode
Gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order2O3And (3) polishing the surface of the powder, then respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally drying the powder, modifying 5 mu L of 0.08mg/mL protein/temperature-sensitive sodium alginate micelle on the surface of the glassy carbon electrode, and drying the modified electrode at room temperature to obtain the protein/temperature-sensitive sodium alginate micelle modified electrode.
4) Preparation of protein molecular imprinting electrochemical sensor
Soaking the dried protein/temperature-sensitive sodium alginate micelle modified electrode in 0.2mg/mL CaCl2In the aqueous solution, Ca is utilized2+Fixing the binding site of the template protein and the temperature sensitive sodium alginate under the cross-linking action of the sodium alginate, soaking for 2h at 40 ℃, and then drying in an oven at 35 ℃ for 30 h; placing in eluent (V) at room temperatureMethanol:VAcetic acidAnd (4) eluting and removing the template protein, and drying at room temperature to obtain the protein molecular imprinting electrochemical sensor.
Example 3
The preparation method of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle comprises the following steps:
(1) preparation of thermo-sensitive sodium alginate
Placing 2g of sodium alginate in a three-neck flask containing 100mL of distilled water, stirring and dissolving at room temperature, and introducing nitrogen gas to remove oxygen dissolved in the solvent; adding 0.5g of ammonium ceric nitrate and 5g N-isopropyl acrylamide monomer, heating to 40 ℃ in a water bath, and carrying out a closed reaction for 3 hours; and after the reaction is finished, standing, naturally cooling, purifying and drying to obtain the thermo-sensitive sodium alginate.
(2) Preparation of protein/temperature-sensitive sodium alginate micelle
Dissolving temperature-sensitive sodium alginate in distilled water to prepare 0.1mg/mL solution, taking 1mL, dropwise adding 100 mu L of 0.1mg/mL bovine serum albumin solution, and self-assembling the protein/temperature-sensitive sodium alginate micelle through weak interaction force between the two under magnetic stirring.
(3) Preparation of protein/temperature-sensitive sodium alginate micelle modified electrode
Gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order2O3And (3) polishing the surface of the powder, then respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally drying the powder, modifying 5 mu L of 0.1mg/mL protein/temperature-sensitive sodium alginate micelle on the surface of the gold electrode, and drying the modified electrode at room temperature to obtain the protein/temperature-sensitive sodium alginate micelle modified electrode.
(4) Preparation of protein molecular imprinting electrochemical sensor
Soaking the dried protein/temperature-sensitive sodium alginate micelle modified electrode in 1.0mg/mL CaCl2In the aqueous solution, Ca is utilized2+Fixing the binding site of the template protein and the temperature sensitive sodium alginate under the cross-linking action of the sodium alginate, soaking for 1h at 40 ℃, and then drying in an oven at 45 ℃; placing in eluent (V) at room temperatureMethanol:VAcetic acidAnd (1: 9) eluting to remove the template protein, and drying at room temperature to obtain the protein molecular imprinting electrochemical sensor.
Example 4
The preparation method of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle comprises the following steps:
(1) preparation of thermo-sensitive sodium alginate
Placing 2g of sodium alginate in a three-neck flask containing 100mL of distilled water, stirring and dissolving at room temperature, and introducing nitrogen gas to remove oxygen dissolved in the solvent; adding 0.5g of ammonium ceric nitrate and 5g N-isopropyl acrylamide monomer, heating to 40 ℃ in a water bath, and carrying out closed reaction for 2 hours; and after the reaction is finished, standing, naturally cooling, purifying and drying to obtain the thermo-sensitive sodium alginate.
(2) Preparation of protein/temperature-sensitive sodium alginate micelle
Dissolving temperature-sensitive sodium alginate in distilled water to prepare 0.5mg/mL solution, taking 1mL, dropwise adding 100 mu L of 0.5mg/mL bovine serum albumin solution, and self-assembling the protein/temperature-sensitive sodium alginate micelle through weak interaction force between the two under magnetic stirring.
(3) Preparation of protein/temperature-sensitive sodium alginate micelle modified electrode
Gold electrode was coated with 0.3 μm and 0.05 μm of α -Al in this order2O3And (3) polishing the surface of the powder, respectively cleaning the powder on an ultrasonic cleaning machine by using distilled water and ethanol, naturally drying the powder, dripping 5 mu L of 0.02mg/mL protein/temperature-sensitive sodium alginate micelle on the surface of the gold electrode, and drying the gold electrode at room temperature to obtain the protein/temperature-sensitive sodium alginate micelle modified electrode.
(4) Preparation of protein molecular imprinting electrochemical sensor
Soaking the dried protein/temperature-sensitive sodium alginate micelle modified electrode in 1.5mg/mL CaCl2In the aqueous solution, Ca is utilized2+Fixing the binding site of the template protein and the temperature sensitive sodium alginate under the cross-linking action of the sodium alginate, and drying in an oven at 30 ℃ after 2 h; placing in eluent (V) at room temperatureMethanol:V Acetic acidAnd (1: 8) eluting to remove the template protein, and drying at room temperature to obtain the protein molecular imprinting electrochemical sensor.
Claims (10)
1. The preparation method of the protein molecular imprinting electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle is characterized by comprising the following steps:
1) preparing temperature-sensitive sodium alginate;
2) preparing a protein/temperature sensitive sodium alginate micelle;
3) a protein/temperature sensitive sodium alginate micelle modified electrode;
4) and (3) preparing the protein molecular imprinting electrochemical sensor.
2. The preparation method of claim 1, wherein in the step 1), the method for preparing the temperature-sensitive sodium alginate comprises the following steps:
placing sodium alginate in water, stirring for dissolving, removing oxygen in water, adding initiator and N-isopropyl acrylamide monomer, and sealing for reaction; and after the reaction is finished, standing, naturally cooling, purifying and drying to obtain the thermo-sensitive sodium alginate.
3. The production method according to claim 1 or 2, wherein in step 1), the initiator is cerium ammonium nitrate, azobisisobutyronitrile, or potassium persulfate.
4. The process according to claim 1 or 2, wherein in step 1), the ratio of the amount of initiator to the amount of substance of the sodium alginate repeating unit is from 1:20 to 1: 2; the mass ratio of the sodium alginate repeating unit to the N-isopropyl acrylamide monomer is 1:10-10: 1.
5. The method according to claim 1 or 2, wherein the reaction is carried out at 10-80 ℃ for 1-15h in step 1).
6. The preparation method according to claim 1, wherein the step 2) is specifically: dissolving the temperature-sensitive sodium alginate prepared in the step 1) in water to obtain a temperature-sensitive sodium alginate solution, dropwise adding a phosphate buffer solution of protein into the temperature-sensitive sodium alginate solution, magnetically stirring, and self-assembling the protein/temperature-sensitive sodium alginate micelle through weak interaction force between the protein and the phosphate buffer solution.
7. The preparation method according to claim 1, wherein the step 3) is specifically: and modifying the protein/temperature sensitive sodium alginate micelle solution on the surface of the electrode, and drying to obtain the protein/temperature sensitive sodium alginate micelle modified electrode.
8. The preparation method according to claim 1, wherein the step 4) is specifically: soaking the modified electrode prepared in the step 3) in a calcium ion solution, taking out and drying; eluting to remove the template protein, and drying at room temperature to obtain the protein molecular imprinting electrochemical sensor.
9. A protein molecular imprinting electrochemical sensor based on a temperature-sensitive sodium alginate self-assembled micelle prepared by the preparation method of any one of claims 1-8.
10. The application of the protein molecularly imprinted electrochemical sensor based on the temperature-sensitive sodium alginate self-assembled micelle prepared by the preparation method of any one of claims 1 to 8 is characterized in that the protein molecularly imprinted electrochemical sensor is used for detecting protein.
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